Abstract

Polyhydroxybutyrate (PHB) is a member of a class of thermoelastic polymers called polyhydroxyalkanoates that serve many bacteria as intracellular storage molecules for carbon and energy. Transgenic plants provide a potential means of producing this polymer cost-effectively. To date, however, few reports of the successful production of this polymer have been published, with the exception of work with transgenic Arabidopsis. Using a variety of chimeric constructs, we have determined that the constitutive, chloroplast-localized expression of one of the genes involved in PHB production-the beta-ketothiolase (phbA) gene-is detrimental to the efficient production of transgenic PHB. The alternate use of either inducible or somatically activated promoters allowed the construction of transgenic PHB-producing potato (Solanum tuberosum) and tobacco (Nicotiana tabacum) plants, although the amount of PHB formed was still rather low. Taking advantage of an inducible promoter, the maximal amount of PHB produced in transgenic potato was 0.09 mg g(-1) dry weight. In transgenic tobacco using a somatically activated promoter, up to 3.2 mg g(-1) dry weight was accumulated. In Arabidopsis, the formation of high levels of PHB had previously been shown to be accompanied by severe negative effects on growth and development of the plant. Phasins are proteins known from PHB-producing bacteria speculated to serve as protectants against the highly hydrophobic surface of the PHB granules in the bacterial intracellular milieu. Co-expression of the phasin gene in parallel with the PHB synthesis genes, however, did not lead to reduced symptom development.

Callus initiation in tobacco leaves 4 weeks after A. tumefaciens-mediated transformation using the constructs pBI-TP-Thio (left), pBI-TP-Red (center), and pBI-TP-Syn (right), which lead to the expression of the genes phbA (β-ketothiolase), phbB (acetoacetyl-CoA reductase), and phbC (PHB synthase), respectively. Callus development and number of regenerated plants increases from left to right.

PHB accumulation in transgenic potato prpABC lines. The amount of 3-HB per gram fresh weight was measured in mature leaves by GC-MS analysis as described in “Material and Methods.” Stprp21+, Stprp3+, and wt+ refer to lines sprayed with 1 mm salicylic acid once a day. Stprp21− and Stprp3− are nontreated controls.

PHB accumulation in transgenic Arabidopsis prpABC lines. The figure shows the same set of plants before induction with salicylic acid (left side) and after 18 d of daily spraying with 1 mm salicylic acid (right side). The three plants at the bottom are wild type. The given values for the accumulation of 3-hydroxybutyrate were determined by GC-MS. They are presented as milligrams per gram fresh weight.